Evidence of a functional receptor
in Prostate cancer cells (LnCaP)
By: Saphir Niakadie
Mentor: Dr. Anthony DePass
Long Island University, Brooklyn Campus
Introduction
• Apoptosis is a
naturally
occurring event
that can be
triggered by a
number of stimuli
such as toxins,
oxidative stress,
calcium influxes
and the activation
of death
receptors.
Introduction con’t
• It has been concluded
that apoptosis is
orchestrated by
calcium levels
(Mattson, 2003), and
we therefore have
hypothesized that
during different stages
of apoptosis, calcium
levels within LnCaP
cells should be
depleted and external
levels should increase
Introduction con’t
• During the course of the summer, I will be working along
side Dr. Anthony DePass and Carleta Williams investigating
the role of ryanodine receptor in prostate cancer (LnCaP)
cells that has been infected with Adenovirus MDA-7 to
induce apoptosis.
Background Information
• Ryanodine receptors are a family of intracellular
calcium channels that aid in the regulation of
calcium levels inside cells.
• There are three known isoforms of the Ryanodine
receptor:
1.RyR1 found in Skeletal muscle
2.RyR2 found in cardiac muscle
3.RyR3 found in brain tissue
Background Information
• Melanoma differentiated associated gene-7 (MDA-7) is a
tumor supressor gene. Studies have shown that transfer
using an adenovirus exhibits minimal damage to normal
cells.
Methods and Procedure
• In order to establish evidence that the
ryanodine receptor is present in LnCaP cells
after treatment with Ad. MDA-7, RT-PCR
and Western Blot analysis of samples
extracted at various time points will be
assessed.
• Calcium measurements will also be taken
from cells infected with the virus.
Methods and Procedures
Adenovirus infection and RNA extraction
• LnCaP cells were cultured in
RPMI medium and
supplemented with 5% CO2.
• Cells were plated in 100mm
petri dishes and infected
with Ad. MDA-7.
• RNA was were extracted
from cells, 24, 48 and 72
hours after initial infection
Methods and Procedures
RT-PCR
• Reverse Transcriptase –
Polymerase Chain Reactions
(RT-PCR) will be done to show
evidence of a functional
ryanodine receptor from mRNA
extracts.
• RT takes the RNA and
transcribes it into DNA, then
PCR uses the very small
segment of DNA and make
millions of copies.
Methods and Procedures
Protein assay and Western Blot analysis
• Protein was also extracted
from LnCaP cells after 24,
48 and 72 hours from initial
infection.
• A polyacrylamide gel will be
used in the western blotting
procedure, then transferred
to a nitrocellulose
membrane for easier
handling.
Discussion
• A previous study has shown that when using RT-PCR two isoforms
RyR1 and RyR2 of the ryanodine receptor were detected in LnCaP cells
(Mariot et al, 2000).
Expected results
• For the RT-PCR procedure we expect an amplified product
of about 554 base pairs (bp) and 476 bp indicating the
ryanodine receptor is fully functioning.
Expected results
• For the Western Blot procedure, we expect to find bands around the 565
kDa marker. This will prove that ryanodine express proteins in LnCaP
cells which then shows evidence that the receptor is existent.
References
•
Mark P Mattson and Sic L .Chan 2003. Calcium orchestrates apoptosis. Nature
cell biology volume 5 number 12
•
Pascal Mariot and Natalia Prevarskya et al 2000. Evidence of functional
Ryanodine Receptor Involved in Apoptosis of Prostate cells. The Prostate
43:205-214
•
Zhaozhong Su and Luni Emdad et al 2005. Unique Aspect of Mda7/IL-24
antitumor bystander activity : establishing a role for secretion of MDA7/IL24protein by normal cells.
http://medix.marshall.edu/~sergeev/images/apoptosis.jpg
http://bricker.tcnj.edu/tech/ltle1/plus.jpg
http://www.scbt.com/
•
•
•
Acknowlegdments
•
•
•
•
•
Dr. Anthony DePass
Carleta Williams
Dr. Sat
Harlem Children Society
MSKCC
THANK YOU!!!